<p>[¹¹C]Carbon monoxide in low concentrations has been used in palladium- or seleniummediated carbonylation reactions such as the synthesis of ¹¹C-imides, hydrazides, amides, carboxylic acids, esters, carbothioates, ketones and carbamoyl compounds.</p><p>In these reactions aryl iodides have been used in most cases. However, less reactive aryl triflate, chloride and bromides were activated using tetrabutylammonium iodide.</p><p>The reactivities of nucleophiles may have influence on the radiochemical yield of the ¹¹Clabelled compounds. Carboxyamination of aryl halides using aniline derivatives yielded 10% of the corresponding ¹¹C-amide. However, the radiochemical yields increased significantly when the aniline derivatives were treated with lithium bis(trimethylsilyl)amide. In contrast, this reagent did not improve the radiochemical yields when primary amines such as methylamine and benzylamine were used. In these cases the radiochemical yields were improved by using pempidine.</p><p>¹¹C-Esterification usually gave low yields. However, the radiochemical yields of ¹¹C-esters could be improved by using magnesium bromide and pempidine.</p><p>An excess of ligand may have a significant impact on palladium-promoted carbonylation reaction. The radiochemical yields of ¹¹C-ketones were improved when using excess amounts of tri-o-tolylphosphine.</p><p>(¹³C)Carbon monoxide may be utilized for the synthesis of ¹³C-substituated compounds in order to confirm the position of ¹¹C-labelling.</p>

<p>Aspartyl proteases can generally be inhibited by peptide mimics containing an uncleavable peptide bond isostere at the proposed cleavage site. One such peptide bond isostere is the hydroxyethylamine moiety, which in this thesis has successfully been incorporated in potential inhibitors of the HIV-1-protease as well as the malarial proteases plasmepsin I and II.</p><p>The human immunodeficiency virus (HIV) has during the last 20 years given rise to a new fast-spread epidemic. The virus protease is one of the foremost targets for drug intervention. In an attempt to improve an earlier design, a P1'-anthranilic acid was exchanged for all four isomers of 2-aminocyclopentanecarboxylic acid, which were synthesized from racemic starting materials, the <i>trans</i> isomers via a novel synthetic route. None of the isomers enhanced potency as compared to the anthranilic acid.</p><p>Because of increasing development of resistance, the pharmaceutical intervention with malaria is becoming rapidly more difficult. A prominent new target for drug research is the hemoglobin degradation pathway. Two of the many proteases involved in this pathway are plasmepsin I and II. Two series of peptide mimics with the hydroxyethylamine were prepared and tested against these enzymes as well as against the similar human protease cathepsin D.</p><p>In the first series the central nitrogen of the target compounds is a secondary amine, derived from natural and unnatural amino acids, the side-chain of which was to bind in the S1'-site of the proteases. It was found that <i>para</i>-aryl substituted phenylalanines resulted in the most active inhibitors. While the P1- and P2-side-chains were kept constant at benzyl and isopropyl respectively, the P3 capping carboxylic acid was varied with a set of diverse carboxylic acids. It was found that many of the carboxylic acids were acceptable.</p><p>A selection of compounds was tested for inhibition of parasite growth in infected human erythrocytes and found to be active.</p><p>In the target compounds of the second series the P1'-side-chain was moved from the α-carbon of the initial amino acids to the adjacent nitrogen, thus rendering this a tertiary amine. The SAR of these compounds suggests that this side-chain cannot be larger than benzyl, which is in sharp contrast to the first series, where both isomers of phenylalanine (i.e. a benzyl group on the α-carbon) render inactive compounds.</p><p>Most of the compounds show a good degree of selectivity for the plasmepsins over cathepsin D, even though a few good inhibitors of the human enzyme could be identified also.</p>

<p>Electrospray ionisation (ESI) coupled to mass spectrometry (MS) is one of the most important detection techniques for chemical analysis of small drugs as well as large biomolecules in life science today. In this thesis, aspects on improved compatibility between liquid based separation systems and mass spectrometric detection were investigated regarding buffers, sample preparation and analysis of polar compounds as well as peptides and protein digests for enhanced ESI-MS performance. </p><p>Capillary electrophoresis (CE) coupled to ESI-MS detection, was evaluated using both a sheath flow interface and a sheathless design. The separation of peptides and small, polar compounds was optimised for both CE-ESI interfaces. The effect of sheath liquid composition was also studied with the aim to improve sensitivity in the ESI-MS detection. </p><p>Polar compounds were retained and separated by capillary ion-pair chromatography coupled to ESI-MS detection. Since commonly used ion-pairing reagents are detrimental to the ESI process they were effectively removed before the ionisation by the use of a trapping column after the separation. Alternatively, the ion-pairing reagents were exchanged to volatile constituents. </p><p>A method for peptide mapping by liquid chromatography (LC)-ESI-MS was developed for lactate dehydrogenase. The method was further enhanced to involve the proteolysis on-line to the LC-ESI-MS. No manual sample handling was then needed and the total analysis time decreased from 7 to 1.5 hours. The amount of information was also shown to increase in the on-line system.</p><p>Finally, the on-line concept was extended to an innovative interface for direct coupling of a pumped liquid flow to an electroosmotically driven flow. This provided a valve-free sample transfer between capillary LC and CE, aiming towards increased peak capacity per unit time for the analysis of complex peptide samples. </p>

<p>Carbon-carbon bond formation reactions are among the most important processes in chemistry, as they represent key steps in the synthesis of more complex molecules from simple precursors. This thesis describes mainly the development of novel regioselective applications of the mild and versatile palladium-catalyzed carbon-carbon coupling method, commonly known as the Heck reaction. In addition, this thesis will focus on environmentally friendly developments of the Heck reaction.</p><p>Novel ligand-controlled internal Heck vinylations of vinyl ethers and enamides to form branched electron-rich dienes were performed with high regioselectivity. The vinylation of 2-hydroxyethyl vinyl ether permits a chemoselective transformation of a vinylic triflate or bromide into a blocked α,β-unsaturated methyl ketone. Furthermore, a simple separation of the palladium catalyst was achieved with new fluorous-tagged bidentate ligands in combination with fluorous solid phase extraction. The reaction times could be reduced up to 1000 times with controlled microwave heating in the palladium-catalyzed reactions with, in the majority of cases, retained, high selectivity. </p><p>The development of a “green” regioselective arylation and vinylation method relying on an aqueous DMF-potassium carbonate system and excluding the toxic thallium salt has been accomplished. Ionic liquids as the versatile and environmentally friendly class of solvents have been used in rapid phosphine-free terminal Heck arylations with controlled microwave heating. Recycling of the catalytic medium was achieved after a simple product purification.</p>

<p>The analysis of pesticide residues in fruit, vegetables, rape seed and water has been improved using developments in sample handling and analytical techniques. The method development is associated with analytical difficulties, since pesticides currently used in agriculture represent a variety of chemical classes having very different physico chemical properties. The method development also encounters difficulties when many various commodity classes with different characteristics are studied. The main task in pesticide residue analysis has been to provide multi residue methods, and traditionally GC has been the main analytical technique.</p><p>In order to regulate the use of hazardous pesticides, the EU commission introduces strict maximum residue levels (MRL). The need for improved sample handling and detection techniques are, however, high due to handling of lower detection limits, complex matrices and the need of more efficient sample throughput. Of the new techniques introduced as alternative techniques to the traditional extraction techniques, pressurised fluid extraction (PFE) has shown to be a promising technique in analysis of pesticide residues in fatty foodstuffs.</p><p>In water analysis, large sample volumes are needed due to low MRLs. The solid phase extraction (SPE) technique allows a concentration of large sample volumes and simplifies the tedious laboratory work with traditional separation funnels. A new approach was to use non-polar solvents for the sample extraction from the earlier used polymeric column. Both these techniques provide low solvent consumption, short extraction times and ability to automate the manual steps. </p><p>An LC-MS/MS multi residue method was finally developed for pesticide residues in fruit and vegetables. The technique is robust and sensitive and allows a simultaneous determination of 57 pesticides and metabolites in one single analysis and without any clean-up steps. The sensitivity was improved to achieve the maximum residue limits needed by EU. Several multi step methods, which involve more costly analysis, has been replaced by this technique.</p>

<p>This thesis exemplifies the strength in the combination of inductively coupled plasma (ICP) spectrometry and electrospray ionization tandem mass spectrometry ESI-MS/MS as detection techniques for liquid chromatography (LC) in the search for and investigation of compounds that bind or can bind hetero-atoms. Furthermore, some aspects involved in the coupling of LC and ICP spectrometry and quantification without identical standards have been studied.</p><p>The importance of using a separation step in combination with ICP spectrometry was shown for urine and blood plasma samples from patients treated with boron neutron capture therapy. In addition to the carrier molecule used in the therapy, one major and a few possible minor metabolites were found in the urine samples. One fragment mass of the major metabolite was obtained with LC-ESI-MS/MS. Liquid chromatography coupled to ICP-MS was also shown to be a valuable tool for fingerprinting metal-binding compounds in complex matrices, such as siderophores (iron-complexing compounds) in soil. The presence of at least two siderophores in a field soil solution sample could be revealed by LC-ICP-MS. Their identities could thereafter be determined by LC-ESI-MS/MS. </p><p>The non-UV-absorbing <i>o</i>-carboranylalanine could be quantified in relation to its degradation products by LC-ICP-AES, which provided information about the mechanism behind the degradation. Moreover, LC-ICP spectrometry was shown to provide an accurate quantification of biomolecules (bias < 10 %) when evaluated from external calibration graphs based on inorganic elemental standards. </p><p>Finally, the causes of the large decrease in boron signal seen when adding acetonitrile to the LC mobile phase in LC-ICP-MS was investigated in some detail. Space charge effects might explain a large part of the depression from carbon species on the boron signal.</p>

<p>Liquid chromatography-mass spectrometry (LC-MS) is a powerful technique for the analysis of endogenous compounds. The introduction of electrospray ionization (ESI) as an interface between LC and MS has contributed strongly to a trend towards miniaturization of LC, due to the possibility to perform ESI at low flow rates. In this thesis, several aspects regarding the design of miniaturized LC systems and electrospray emitters were investigated. In addition miniaturized LC-ESI-MS have been used for the qualitative and quantitative analysis of endogenous polar compounds, peptides and protein digests. </p><p>The performance of miniaturized LC-MS was compared using different electrospray emitter configurations. The results indicated that the efficiency of the LC system is rather independent of the configuration of the emitter. </p><p>The lifetime of gold-coated fused silica electrospray emitters based on vapor deposited adhesion layers of titanium were investigated. The long lifetime of the emitter facilitates the use in LC-MS experiments, exemplified LC-MS by analysis of neuropeptides.</p><p>The ESI voltage is shown to interfere with liquid chromatographic separations performed in packed porous graphitic carbon capillary column. This interference is ascribed to the presence of an electric field over the conductive column in absence of a ground point between the column and the ESI emitter.</p><p>The solid supported enhanced microdialysis for analysis of neuropeptides were compared with conventional microdialysis. The difference between the two methodologies were evaluated by LC-MS analysis of the microdialysates. The solid supported method gave in general higher relative recoveries.</p><p>Finally, a method of standard addition was developed to determine total level of tryptophan and two of its metabolites in human plasma by capillary LC-ESI tandem mass spectrometry. The method was applied in a clinical study of multiple scleroses patients treated with cytokines (IFN Beta 1a, 1b). The results show that the intervention effects the tryptophan metabolism.</p>

<p>This thesis describes free-radical reactions for the construction of tetrahydrofuran and pyrrolidine derivatives. The studies are concerned with (<i>i</i>) diastereoselectivity in radical cyclisation, (<i>ii</i>) construction of tetrahydrofuran-3-ones and pyrrolidin-3-ones <i>via</i> radical carbonylation/cyclisation and (<i>iii</i>) synthesis of tetrahydrofuran derivatives <i>via</i> group-transfer cyclisation of organochalcogen compounds.</p><p>(<i>i</i>) Diastereoselectivity in the synthesis of tetrahydrofuran derivatives <i>via</i> radical cyclisation was controlled by addition of Lewis acids. In the synthesis of 2,4-disubstitued tetrahydrofurans, the <i>trans</i>-isomer was formed as the major product in the unperturbed reaction. Upon addition of trialkylalumiums the diastereoselectivity was reversed. In a similar fashion, <i>exo</i>/<i>endo</i>-diastereoselectivity in the synthesis of bicyclic 2,3,4-trisubstituted tetrahydrofurans could also be controlled.</p><p>(<i>ii</i>) Procedures for construction of tetrahydrofuran-3-ones and pyrrolidin-3-ones were presented. Epoxides were ring-opened with benzeneselenolate or benzenetellurolate and the resulting <i>β</i>-hydroxyalkyl phenyl chalcogenides were vinylated using ethyl propiolate/NMM or <i>E</i>-1,2-bis(phenylsulfonyl)ethylene/NaH. The corresponding nitrogen analogues were accessed by <i>N</i>-vinylation of aziridines followed by benzeneselenolate ring-opening. The two types of organochalcogen radical precursors were then treated with TTMSS/AIBN under an atmosphere of carbon monoxide (80 atm) to afford tetrahydrofuran-3-ones and pyrrolidin-3-ones, respectively, in high yields.</p><p>(<i>iii) </i>Microwaves were found to induce group-transfer cyclisation of <i>β</i>-allyloxyalkyl aryl chalcogenides. Short time heating (3-10 min) at 250 ^oC in ethylene glycol was required to obtain tetrahydrofuran derivatives in 60-91% yield.</p>

This thesis is divided into two parts based on a total of 8 papers: Part 1: Synthesis, physicochemical and biochemical studies of chemically modified oligonucleotides and their duplexes and triplexes. Potency of the chromophore conjugated DNA oligonucleotides as antigene and antisense gene repressors was evaluated. The effect of geometry, bulk and ¥ð-electron density of a series of chromophores, tethered at the 5'-end of oligonucleotides, as well as the effect of the linker nature, length and the attachment site of the chromophore to the oligo were explored based on the stability of the duplexes and triplexes. A dramatic improvement in the triplex stability with ara-U linked phenazine oligo (potent antigene) was achieved (¥ÄTm = 16.5¢ª C). A number of selected phenazine and dipyridophenazine tethered antisense oligos (AONs) and their phosphorothioate analogues were shown to form the AON/RNA hybrid duplexes with enhanced thermal stability. CD experiments revealed that these duplexes have the global structure unaltered from that of the native counterpart. RNase H degradation studies on three RNA targets having different degrees of folded structures showed that tethering of phenazine and dipyridophenazine increases the hydrolysis rates (potent antisense) of the target RNA, and that chemical nature of the chromophore influences the RNase H cleavage pattern. Further investigation at the RNA saturated conditions revealed that 3'-tethered chromophores influence the substrate recognition, and the kinetics of the cleavage by RNase H. Conjugation of different chromophores, charged polyaromatic systems and metal complexes with polyaromatic ligands at different sites of the AON revealed that RNase H is very sensitive to any modifications in the middle region of the AON/RNA duplex. On the contrary, any modification at the 3'-end of the AON regardless of the bulk of the substituent or presence of positive charge can be easily tolerated by the enzyme. Sensitivity of the RNase H towards the local structural changes in the AON/RNA hybrid was probed with a number of AONs containing a single 1-(1',3'-O-anhydro-©¬-<u>D</u>-psicofuranosyl)thymine with locked 3'-endo sugar conformation at different sites of AON. RNase H degradation studies revealed that the local conformational changes brought by the constrained nucleoside, although invisible by CD, span in the hybrid as far as 5 nucleotides toward the 5'-end of the AONs (3'-end of RNA), showing the unique transmission of the structural distortion from a single modification site. The results also showed that the structural requirements for the substrate binding and substrate cleavage by RNase H appear to be different. Part 2: Preparation of biologically important isotope labelled oligo-RNAs for the NMR structure determination in solution. Synthesis of the non-uniformly 13C5 labelled 29mer HIV-1 TAR RNA was achieved by solid-phase synthesis using 13C5 labelled ribonucleosides from 13C6-<u>D</u>-glucose). Two hammerhead forming RNAs (16mer and 25mer) were synthesized according to the Uppsala NMR-window strategy, where the sugar residues of the nucleosides forming stem I, II and the loop of the stem III of the resulting hammerhead complex were deuterated. UV melting and high resolution NMR structural studies showed that the 16mer RNA under quasiphysiological condition folds to a very stable hairpin structure, which prevents formation of a hammerhead RNA with the 25mer, primarily owing to thermodynamic reasons.

Capillary electroseparation methods<b> </b>are exciting new techniques with very broad application areas and vast potential in pharmaceutical and biomedical analysis. To improve the limit of detection (LOD) capillary zone electrophoresis (CZE) has been combined with isotachophoretic (ITP) preconcentration in a single capillary. Using the ITP-CZE combination the LOD can be improved at least 100-fold. Laser-induced fluorescence (LIF) detection is more sensitive and more selective than the most common detection technique, UV, and the intensity and focusing capability of LIF fits well with the small dimensions in CZE. The total sensitivity enhancement attained for a new acetylcholinesterase inhibitor, NXX-066, by using ITP-CZE-LIF was more than 5500-fold compared to CZE-UV. Capillary electrochromatography (CEC) combines the high separation efficiency of CZE with the vast possibilities to improve selectivity of HPLC. We have examined different ways to solve the problem of extensively tailing peaks and studied the influence of the mobile phase composition on the electrochromatographic performance for a number of tricyclic antidepressants and related quaternary ammonium compounds. (1) Adding aliphatic amines to the mobile phase in reversed phase CEC. The effect on the chromatographic performance was coupled to the hydrophobicity of the additive and the amine of our choice was dimethyloctylamine. (2) Silica-based cation exchangers with different pore sizes. The large-pore materials promoted pore flow, but this had no positive influence on the performance. The small-pore (highest surface area) particles gave the best selectivity. (3) Designing special continuous beds. As the bed is covalently attached to the capillary wall, problems related to retaining frits are avoided. The stationary phase most suitable for our analytes had a molar ratio of 1:80 between the functional ligands, vinyl sulphonic acid and isopropyl groups, respectively. The LOD was lowered 26000-fold by dissolving the sample in a low-conducting medium.